Prem L. Sharma

Nucleoside Inhibitors of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

The development of novel compounds that can effectively inhibit both wild type and the most consensus resistant strains of human immunodeficiency virus type 1 (HIV-1) is the primary focus in HIV disease management. Combination therapy, comprising at least three classes of drugs, has become the standard of care for acquired immunodeficiency syndrome (AIDS) or HIV-infected individuals. The drug cocktail can comprise all three classes of HIV inhibitors, including nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Due to their competitive mode of inhibition and requirement for metabolic activation, almost all NRTI drugs lack the virological potency of NNRTI or PI drugs. However, data from clinical trials indicate that sustained viral suppression could not be achieved with NRTI, NNRTI or PIs alone. Therefore, the NRTIs will remain essential components of highly active antiretroviral therapy (HAART) for the foreseeable future, because they enhance the virological potency of the regimen, they do not bind excessively to protein and most regimens are small pills/tablets given once a day. It has become apparent in recent years that the prolonged use of certain NRTIs exhibits adverse events as a class, limiting the length of time for which they can be safely used. Of major clinical concern is their association with the potentially fatal lactic acidaemia and hepatic steatosis. These class events, as well as individual drug effects, such as peripheral neuropathy, are linked to delayed mitochondrial destruction. In addition to toxicity, the development of resistance-conferring mutations against exposure to nucleoside analogs currently in use influences long-term therapeutic benefits. Of critical importance for the evaluation of new NRTIs are recent studies showing that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway.

Characterization of an acyclovir-resistant herpes simplex virus type 2 strain isolated from a premature neonate.

Acyclovir resistance is not a recognized problem among neonates with perinatal herpes simplex virus (HSV) infection. A premature newborn with neurocutaneous HSV infection was treated for 21 days with acyclovir. Disseminated disease recurred 8 days later. A recurrent isolate was resistant to acyclovir and lacked thymidine kinase activity on the basis of a frameshift mutation in the thymidine kinase (tk) gene. Compared with the sensitive isolate obtained during primary infection, replication of the resistant isolate was reduced on primary and permanent cells and even further impaired on cells deleted for cellular tk. The resistant isolate lacked virulence in a murine model of genital infection. Acyclovir-resistant HSV-2 mutants can develop rapidly in neonatal infection and cause clinically significant disease, despite decreased replication in vitro and attenuated virulence in an animal model.

Murine and simian retrovirus models: the threshold hypothesis

By considering the dynamic relationship between retroviruses and their hosts, we have developed a unifying hypothesis to explain such disparate clinical phenomena as differential pathogenicity of a given virus in adults and neonates, transient infection with clearance of provirus-containing cells, long-term non-progression and vaccine effects of fully pathogenic viruses. The threshold hypothesis predicts that an opportunity exists during acute retroviral infection to influence the ultimate clinical outcome: if virus replication is kept below threshold by any means, including drug therapy or passive immunoprophylaxis with neutralizing antibodies, the host will prevail and win the race.

Quantifying mixed populations of drug-resistant human immunodeficiency virus type 1.

ABSTRACT In order to survive prolonged treatment with antiretroviral nucleoside analogs, the human immunodeficiency virus type 1 (HIV-1) is selectively forced to acquire mutations in the reverse transcriptase (RT) gene. Some of these mutations are more common than others and have become markers for antiretroviral resistance. For the early detection of these markers, a novel MultiCode-RTx one-step testing system to rapidly and simultaneously characterize mixtures of HIV-1 targets was designed. For cDNA, nucleotide polymorphisms for codon M184V (ATG to GTG) and K65R (AAA to AGA) could be differentiated and quantified even when the population mixture varied as much as 1 to 10,000. Standard mixed-population curves using 1 to 100% of the mutant or wild type generated over 4 logs of total viral particle input did not affect the overall curves, making the method robust. The system was also applied to a small set of samples extracted from infected individuals on nucleoside reverse transcriptase inhibitor therapy. Of 13 samples tested, all were positive for HIV and 10 of the 13 genotypes determined were concordant with the line probe assay. MultiCode-RTx could be applied to other drug-selected mutations in the viral genome or for applications where single-base changes in DNA or RNA occur at frequencies reaching 0.01% to 1%, respectively.

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

ABSTRACT The fluorescent dye-labeled dideoxynucleotide automated DNA sequencing system has been routinely used for monitoring the development of resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) and protease genes during therapy. This system has provided information regarding the presence of mixtures of nucleotides in the clinical samples but has not previously been validated for the quantitative determination between peak heights and relative DNA concentration. We evaluated this system by using various ratios of wild-type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced, and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentration demonstrated a statistically significant linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the fluorescent dye-labeled dideoxynucleotide system. These strategies have broader implications for the quantification of replication fitness of viruses, particularly those containing RT mutations at codons 74 and 184.

Comparative Analysis of In Vitro processivity of HIV-1 Reverse Transcriptases Containing Mutations 65R, 74V, 184V and 65R+74V

While HIV-1 reverse transcriptase (RT) mutations of M to V at position 184 are commonly observed in the clinic, the double mutation of 65R+74V is rarely seen. It has been demonstrated that rapid R-->K reversion occurs at RT codon 65 during replication of HIV-1 in human peripheral blood mononuclear cells containing 65R+74V mutations and that processivity of the RT is reduced relative to wild type. However, clinical studies show that M184V can be detected after several months of therapy interruption, suggesting more effective processivity. Herein, the in vitro RT processivity of genetically engineered M184V and double mutant 65R+74V was compared. Virion-associated RTs of WT pNL4-3, K65R, L74V, M184V and 65R+74V were used to perform RT processivity assays in the presence of trap, poly(rC)-oligo(dG). Both RTs with 184V and 65R+74V mutations exhibited similar processivity when compared with each other and a significantly decreased processivity as compared to WT RT. Both mutant RTs synthesized shorter cDNA molecules (37-42 nt) relative to WT RT, which made longer (65-70 nt) cDNA molecules. Since these surprising biochemical results cannot explain the clinical phenotype, a hypothesis is presented to explain the discrepancy and suggest new approaches for future testing.

Retrovirus reverse transcriptases containing a modified YXDD motif

The YXDD motif, where X is a variable amino acid, is highly conserved among various viral RNA-dependent DNA polymerases. Mutations in the YXDD motif can abolish enzymatic activity, alter the processivity and fidelity of enzymes and decrease virus infectivity. This review provides a summary of the significant documented studies on the YXDD motif of HIV-1, simian immunodeficiency virus, feline immunodeficiency virus and murine leukaemia virus and the impact of mutation that this motif has had on viral pathogenesis and drug treatment.

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus

BackgroundThe major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.MethodsProviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.ResultsReplication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.ConclusionsThe improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.

Reverse transcriptase mutation K65N confers a decreased replication capacity to HIV-1 in comparison to K65R due to a decreased RT processivity

In addition to K65R, the other mutation observed at HIV-1 RT codon 65 is K65N. While K65N appears to have a phenotypic effect similar to K65R, it is less frequent during clinical trials. We compared the relative impact of K→N with respect to K→R change on viral replication capacity (RC). Mutant viruses were created and replication kinetics assays were performed in PBM cells. Analysis of RCs revealed a significant loss in replication (p=0.004) for viruses containing K65N mutation in comparison to those with K65R mutation. RT processivity assays showed a significant decrease in the processivity of K65N RT in comparison to K65R RT. We demonstrated that the significant decrease in RC of K65N viruses is related to the impaired RT processivity of K65N RT in comparison to K65R, and that the selection of the K65R mutation may be favored in clinical use of antiretroviral drugs compared to K65N.

Replication-Independent Expression of Anti-Apoptosis Marker Genes in Human Peripheral Blood Mononuclear Cells Infected with the Wild-Type HIV-1 and Reverse Transcriptase Variants

Clinical trials with highly-active antiretroviral therapy (HAART) have shown that a substantial number of patients continue to show a decrease in viral load and/or increase or stable CD4(+) T-cell numbers even in the presence of multidrug resistant (MDR) viruses. We compared replication capacity (RC) and expression of anti-apoptosis marker genes (AAMGs) in human peripheral blood mononuclear (PBM) cells infected with NL4-3 (wild-type; WT) and mutant viruses. Replication kinetics assays showed a significant decrease in RC of all mutant viruses in comparison to the WT virus. The viruses containing patient-derived MDR RT without the K65R mutation (PSD5.2) replicated efficiently in comparison to the viruses with MDR RT containing the K65R mutation (PSD5.1), or the single mutations K65R and M184V. Compared with WT, a significant decrease in RCs of viruses: K65R (RC=0.39±0.02; p≤0.0001), M184V (RC=0.72±0.04; p≤0.0001), PSD5.1 (RC=0.32±0.04; p≤0.0001), and PSD5.2 (RC=0.90±0.04; p=0.002) was observed on day 10. RT-PCR-based apoptosis array was performed on total cellular RNA. Recombinant virus PSD5.2 showed a 1.5- to 6-fold upregulation in 8 AAMGs (AKT1, BAG3, BCL2A1, BFAR, BIRC2, BNIP1, BNIP3, and CFLAR) on day 1 and day 7 post-infection with respect to WT virus. PSD5.1 showed upregulation of only one gene (BAG1) on day 1 (1.75-fold) and day 7 (1.97-fold). Point mutant K65R showed a 1.5- to 4-fold upregulation of six AAMGs on day 7. Viruses with the M184V mutation showed upregulation of only one gene (BAG1). These observations indicate that the upregulation of specific AAMGs may not be dependent on the RCs of HIV-I variants, and that the possible interaction among mutated RT residues and viral and/or host proteins may induce CD4(+) T-cell-protective anti-apoptosis proteins.